DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection.

DExD/H-box helicases are crucial regulators of RNA metabolism and antiviral innate immune responses; however, their role in bacteria-induced inflammation remains unclear. Here, we report that DDX5 interacts with METTL3 and METTL14 to form an m6A writing complex, which adds N6-methyladenosine to transcripts of toll-like receptor (TLR) 2 and TLR4, promoting their decay via YTHDF2-mediated RNA degradation, resulting in reduced expression of TLR2/4. Upon bacterial infection, DDX5 is recruited to Hrd1 at the endoplasmic reticulum in an MyD88-dependent manner and is degraded by the ubiquitin-proteasome pathway. This process disrupts the DDX5 m6A writing complex and halts m6A modification as well as degradation of TLR2/4 mRNAs, thereby promoting the expression of TLR2 and TLR4 and downstream NF-κB activation. The role of DDX5 in regulating inflammation is also validated in vivo, as DDX5- and METTL3-KO mice exhibit enhanced expression of inflammatory cytokines. Our findings show that DDX5 acts as a molecular switch to regulate inflammation during bacterial infection and shed light on mechanisms of quiescent inflammation during homeostasis.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels.

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.

Effects of duration of thermal stress on growth performance, serum oxidative stress indices, the expression and localization of ABCG2 and mitochondria ROS production of skeletal muscle, small intestine and immune organs in broilers.

The purpose of the current study was to investigate that effect of duration of thermal stress on growth performance, oxidative stress indices in serum, the expression and localization of ABCG2, and mitochondria ROS production in skeletal muscle, small intestine and immune organs, and then to further reveal correlations between indicators. At 28 days of age, sixty broilers were randomly divided into the control group (25 ±â€¯2 °C; 24 h/day) and the heat stress group (36 ±â€¯2 °C; 8 h/day lasted for 1 week or 2 weeks). Fifteen broilers per group were respectively euthanized, and some samples were respectively collected from the control and the heat stress groups at the end of the 1st week or the 2nd week of heat stress. A typical heat stress response has been observed at this temperature. Compared with the control group, the birds subjected to heat stress at the end of the 1st week reduced (P ＜ 0.05) body weight (BW), average daily feed intake (ADFI), average daily gain (ADG), the activity of serum antioxidant enzyme and content of glutathione (GSH), while increased (P ＜ 0.05) feed conversion ratio (FCR), serum corticosterone and malondialdehyde (MDA) levels. However, when the heat stress lasted for the end of the 2nd week, there was no significant difference (P ＞ 0.05) in ADFI, ADG, FCR and serum contents of corticosterone, MDA and GSH. Regardless of duration of thermal stress, the localization of ABCG2 protein had no change. Moreover, heat stress also did not affect (P ＞ 0.05) the IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in the pectorales, crureus, duodenum, jejunum, ileum and spleen, while significantly increased (P ＜ 0.05) the corresponding tissues ROS production at the end of the 1st week of heat stress. In contrast, at the end of the 2nd week of heat stress, IOD of the ABCG2 positive portion and the expression of the ABCG2 mRNA in heat stress group significantly increased (P ＜ 0.05), while the corresponding tissues ROS production had no difference (P ＞ 0.05) compared to the control group. Collectively, duration of thermal stress affects growth performance, serum oxidative stress indices, and the expression of ABCG2 and the ROS production of broiler tissues in a time-dependent manner. There is a negative correlation between the expression of ABCG2 and the ROS production in the corresponding tissues under heat stress.